7 replies to this topic

[science noob]

Which is more crucial in terms of protein transfer? Which should ideally be kept constant?

[note1988]

With my experience, I chose 200V as my constant voltage for 2 h. At the same time, I noticed the current was begin with 160~ mA and end at about 200~ mA.  Hope can help you.

[note1988]

note1988, on 22 November 2011 - 10:21 PM, said:

With my experience, I chose 200V as my constant voltage for 2 h. At the same time, I noticed the current was begin with 160~ mA and end at about 200~ mA.  Hope can help you.

Sorry, the voltage was 75V. And I use Bio-rad mini-protean system.

[BioMiha]

I say current. I always tell my students that constant voltage (or potential difference) drives the electrophoretic separation of proteins because you want them to separate according to their electrophoretic mobility. However, when blotting you want ALL the proteins to transfer in which case you want a torrent of ions to drive the transfer process. That is my theory at least. I use 200 mA for 1 h and the BioRad mini protean system as well.

[mdfenko]

i prefer a constant current density (mA/cm²). but it depends on the apparatus and the manufacturer's recommendation.

[science noob]

BioMiha, on 23 November 2011 - 01:10 AM, said:

I say current. I always tell my students that constant voltage (or potential difference) drives the electrophoretic separation of proteins because you want them to separate according to their electrophoretic mobility. However, when blotting you want ALL the proteins to transfer in which case you want a torrent of ions to drive the transfer process. That is my theory at least. I use 200 mA for 1 h and the BioRad mini protean system as well.

200mA with constant voltage? May I ask what voltage were you using for 1 h?

mdfenko, on 23 November 2011 - 09:27 AM, said:

i prefer a constant current density (mA/cm²). but it depends on the apparatus and the manufacturer's recommendation.

Please explain the constant current density? so what is the current for 1 cm²?

[mdfenko]

science noob, on 01 December 2011 - 04:39 AM, said:

Please explain the constant current density? so what is the current for 1 cm²?

the apparatus that i used was for semi-dry transfers. it was originally made and sold by abn then millipore. it had these funky carbon loaded rubber(?) electrode covers. the manual said to transfer with a current of 2.5 mA/cm² for no longer than 1 hour (most transfers were 30-40 minutes).

the point is: read the manual for the apparatus that you use. it will give you conditions that they have determined to be optimal for average usage and useful tweaks that you can try if your results don't suffice.

[PHVS]

I use the XCell SureLock system from Invitrogen and always do my transfers using 30-40V and 250 mA for 1h.